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Northern Blot
The Northern Blot technique was originally designed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University to investigate RNA structure and quantity, though today it is used almost exclusively to quantify gene transcription (using messenger RNA mRNA) and thus assess gene expression (1, 5). The Northern Blot technique was so named based on its similarity to the Southern Blot technique invented 2 years previously in 1975 by Edwin Southern, though it should be noted that Southern blotting analyzes DNA while Northern blotting analyzes RNA so the 2 are inherently very different (1, 2, 4). Northern Blotting is an invaluable technique; the fact that it remains in use 4 decades after its genesis serves as a testament to its auspicious nature. Procedure In order to assess the RNA within a cell or tissue, such RNA must first be isolated. There are multiple ways in which one can extract the entire RNA complement from a collect of cells, including organic extraction and solid matrix extraction methods, but all of these different techniques strive to accomplish the same thing: the removal of contaminating cellular debris, proteins and DNA (5). It is important to also note that it is not always necessary or possible to isolate mRNA from total RNA. The steps outlined below are only an example of a Northern Blot procedure that could be used. Solid Matrix mRNA Isolation In any type of nucleic acid isolation, it is imperative to first centrifuge your cells of interest and then place the pelleted cellular contents obtained in an alkaline solution to facilitate cell lysis. When isolating RNA, this cell lysis step is usually preformed using a detergent or phenol solution with a high salt concentration. In this step, it is essential that the solution being used either contains RNAses inhibitors or inhibits RNAse activity in some other way so that the RNA sample of interest is not degraded. Once this step has been completed, the lysate is treated with a neutralizing, chaotropic (i.e. denaturing) buffer and then added to a commercially provided spin column containing either a specialized resin matrix or silica beads. DNAse may also be added at this step to prevent DNA contamination. If mRNA is to be isolated, columns or beads with imobilized single-stranded thymine or uracil oligomers will be used as these nucleic acid sequences will selectively bind (using hydrogen bonds) to the poly A tails present at the 3' ends of all processed mRNA (5). The column then undergoes a series of washes wth comercially supplied buffers to remove any residual salts, cellular debris or DNA. After this series of washes, the column is then subjected to a warmed, low salt buffer containing detergent (the detergent will break the hydrogen bonds holding the poly A mRNA tail to the polyT or polyU oligomers). The eluent collected from this final wash contains your mRNA (5). Before proceeding to gel electrophoresis, it is critical that you check the purity and concentration of the mRNA (or simply RNA) use have just collected. For this purpose, a Nanodrop is usually employed. Nandrop technology determines your nucleic acid concentration by measuring the absorbance of your sample at different wavelengths (nucleic acids absorb maximally around 260nm). It is up to the researcher to then determine the purity of the sample by looking at the ratios of optical densities at specific wavelengths: 260nm/280nm and 260nm/230nm. As was mentioned previously, nucleic acids absorb light maximally at 260nm. The 280nm reading reflects the amount of protein contamination present in your sample, as protein absorbs maximally around 280nm, while the 230nm reading reflects the amount of phenol, EDTA, or carbohydrates present in your sample. For an RNA isolation, you would want a 260nm/280nm ratio greater than 2 as this would indicate a high concentration of nucleic acids; with mRNA isolation however, it is unlikely that you will have such a ratio. In any type of nucleic acid isolation, the 260nm/280nm ratio should be greater than 1.6 as any number lower than this reflects protein contamination and a ratio between 1.8 and 2 is thought to represent DNA isolation. 260nm/230nm ratios less than 2 indicate phenol, EDTA or carbohydrate contamination. Gel Electrophoresis The isolated sample is then applied to a 0.8-1.5% agarose gel or a polyacrylamide gel. The gel selected should reflect the size of the RNA you are attempting to isolate and quantify: if you are endeavoring to isolate a smaller RNA transcript, it is generally recommended that you use a polyacrylamide gel (5). Whatever type of gel is selected must still contain a denaturing agent, such as urea or formaldehyde, as such an agent is necessary to prevent RNA folding and ensure proper RNA-probe hybridization later on(2, 5). The gel being used may also contain a nucleotide staining gel (such as SyBr green) that will stain the sample directly so that it is possible to visualize the separation of your sample after preforming gel electrophoresis. Gel electrophoresis will separate the contents of the sample by their charge to mass ratios, propelling negatively charged biomolecules to the cathode electrode pole of the gel at different speeds inversely proportional to their sizes. A loading buffer containing a loading dye (usually bromophenol blue), a buffer solution and a density agent that will increase the density of the test sample in relation to the buffer submerging the gel (allowing it to sink into the well of the gel) will be mixed with the sample and the two will be added to the gel wells together. The loading dye will race slightly ahead of the sample during gel electrophoresis, allowing the termination of the electric current at the appropriate time (5). Blotting, Probing and Analysis The RNA that has been separated using gel electrophoresis is then transferred to a nitrocellulose blotting membrane using one of the many transfer methods available (some examples include the electrophoretic transfer and the vacuum transfer techniques) (5). Once the RNA has been successfully transferred to the membrane, single stranded DNA or RNA probes that are complementary to the sequences of interest and are either labeled radioactively or enzymatically are applied to the membrane. Often, probes containing sequences complementary to highly conserved and almost universally present RNA molecules will be added to make sure the experimental conditions have allowed proper hybridization between probes and target sequences. One such probe is a probe complementary to Beta-Actin mRNA, asBeta-Actin is a highly-conserved cytoskeleton protein that is almost ubiquitously present in all human cells. After a period of incubation (and treatment with a specific substrate if the probes are enzymatically labeled), all unbound probes are removed in a series of washes and the membrane is exposed to an X-Ray film. All RNA-Probe hybrids present on the membrane will be visible as discreet bands on the film (3 ,4 ). Uses Over the last 4 decades, Northern blotting has been used to demonstrate variations in gene expression within different tissues and during different developmental stages (2). Northern blotting has been used to measure differential gene expression during periods of environmental stress or throughout the course of various pathologies. This technique has also been used to demonstrated the increase in oncogene transcription and the decrease in tumor suppressor gene expression in malignant cells as compared to normal cells. Northern Blot analyzes have also been used to investigate the mechanisms behind tissue transplant rejection (2). Although most Northern Blot assays are used to investigate gene expression within particular tissues, Northern Blot analyses can also reveal mRNA structural abnormalities resulting from any sort of irregularity during transcription or post-transcriptional processing, such as erroneous splicing (5). Several human diseases, such as Beta-thalassemias and familial isolated growth hormone deficiency, are due to splicing abnormalities and can be detected using Northern Blotting techniques. As Buckingham so correctly observes, "analysis of RNA structure and quantity indirectly reveals mutations in the regulatory or splicing signals in DNA (5)". References Internet 1. History of Northern Blotting 2. Wikipedia Article on Norther blot 3. Northern Blot Procedure 4. Scitable Northern Blot Print 5. Buckingham, L. (2012). ''Molecular diagnostics: Fundamentals, methods, and clinical applications ''. (2nd Ed. ed., pp. 110-111). Philadelphia, PA: F.A Davis Company